The biosynthesis, structure, storage and release of peptides related to ACTH and endorphin will be studied. The immunochemical methods previously used to study ACTH and endorphin biosynthesis in mouse pituitary tumor cells will be used to study ACTH and endorphin biosynthesis in primary cell suspensions prepared from the anterior and intermediate lobes of the rat pituitary. Similar techniques will be used to investigate the biosynthesis ACTH and endorphin in several non-pituitary tissues. Post-translational processing of the pro-ACTH/endorphin molecule in AtT-20 mouse pituitary tumor cells and in rat anterior and intermediate pituitary cell suspensions will be examined following subcellular fractionation to separate rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi vesicles and secretory granules. Particular attention will be directed towards understanding the processes of glycosylation and proteolytic cleavage as they occur in these three different systems. The factors responsible for determining the characteristic set of pro-ACTH/endorphin cleavage products released by intermediate lobe cells, anterior lobe cells and tumor cells will be investigated. The purification of milligram amounts of glycosylated ACTH, beta-lipotropin and a glycopeptide fragment of pro-ACTH/endorphin from tumor cell culture medium will continue. Purified tumor cell molecules will be used for structural studies, in vitro studies of biosynthetic processing, investigation of their effects on rat adrenal cell suspensions and development of new radioimmunoassays. The physiological regulation of ACTH/endorphin synthesis and secretion from anterior and intermediate pituitary cell suspensions will be studied.